Cell Therapy Attenuates Cardiac Dysfunction Post Myocardial Infarction: Effect of Timing, Routes of Injection and a Fibrin Scaffold

Background: Cell therapy approaches for biologic cardiac repair hold great promises, although basic fundamental issues remain poorly understood. In the present study we examined the effects of timing and routes of administration of bone marrow cells (BMC) post-myocardial infarction (MI) and the efficacy of an injectable biopolymer scaffold to improve cardiac cell retention and function. Methodology/Principal Findings: (99m)Tc-labeled BMC (6x10(6) cells) were injected by 4 different routes in adult rats: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV(+)) to mimic coronary injection, and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days post-MI and cell retention was estimated by gamma-emission counting of the organs excised 24 hs after cell injection. IM injection improved cell retention and attenuated cardiac dysfunction, whereas IV, LV or LV* routes were somewhat inefficient (< 1%). Cardiac BMC retention was not influenced by timing except for the IM injection that showed greater cell retention at 7 (16%) vs. 1, 2 or 3 (average of 7%) days post-MI. Cardiac cell retention was further improved by an injectable fibrin scaffold at day 3 post-MI (17 vs. 7%), even though morphometric and function parameters evaluated 4 weeks later displayed similar improvements. Conclusions/Significance: These results show that cells injected post-MI display comparable tissue distribution profile regardless of the route of injection and that there is no time effect for cardiac cell accumulation for injections performed 1 to 3 days post-MI. As expected the IM injection is the most efficient for cardiac cell retention, it can be further improved by co-injection with a fibrin scaffold and it significantly attenuates cardiac dysfunction evaluated 4 weeks post myocardial infarction. These pharmacokinetic data obtained under similar experimental conditions are essential for further development of these novel approaches.

Association of alpha1a-adrenergic receptor polymorphism and blood pressure phenotypes in the Brazilian population

Background: The alpha1A-adrenergic receptor (alpha(1A)-AR) regulates the cardiac and peripheral vascular system through sympathetic activation. Due to its important role in the regulation of vascular tone and blood pressure, we aimed to investigate the association between the Arg347Cys polymorphism in the alpha(1A)-AR gene and blood pressure phenotypes, in a large sample of Brazilians from an urban population. Methods: A total of 1568 individuals were randomly selected from the general population of the Vitoria City metropolitan area. Genetic analysis of the Arg347Cys polymorphism was conducted by polymerase chain reaction/restriction fragment length polymorphism. We have compared cardiovascular risk variables and genotypes using ANOVA, and Chi-square test for univariate comparisons and logistic regression for multivariate comparisons. Results: Association analysis indicated a significant difference between genotype groups with respect to diastolic blood pressure (p = 0.04), but not systolic blood pressure (p = 0.12). In addition, presence of the Cys/Cys genotype was marginally associated with hypertension in our population (p = 0.06). Significant interaction effects were observed between the studied genetic variant, age and physical activity. Presence of the Cys/Cys genotype was associated with hypertension only in individuals with regular physical activity (odds ratio = 1.86; p = 0.03) or younger than 45 years (odds ratio = 1.27; p = 0.04). Conclusion: Physical activity and age may potentially play a role by disclosing the effects of the Cys allele on blood pressure. According to our data it is possible that the Arg347Cys polymorphism can be used as a biomarker to disease risk in a selected group of individuals.

ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser(1270)

Objectives: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra-or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).

Genetic Variants of Diabetes Risk and Incident Cardiovascular Events in Chronic Coronary Artery Disease

Objective: To determine whether information from genetic risk variants for diabetes is associated with cardiovascular events incidence. Methods: From the about 30 known genes associated with diabetes, we genotyped single-nucleotide polymorphisms at the 10 loci most associated with type-2 diabetes in 425 subjects from the MASS-II Study, a randomized study in patients with multi-vessel coronary artery disease. The combined genetic information was evaluated by number of risk alleles for diabetes. Performance of genetic models relative to major cardiovascular events incidence was analyzed through Kaplan-Meier curve comparison and Cox Hazard Models and the discriminatory ability of models was assessed for cardiovascular events by calculating the area under the ROC curve. Results: Genetic information was able to predict 5-year incidence of major cardiovascular events and overall-mortality in non-diabetic individuals, even after adjustment for potential confounders including fasting glycemia. Non-diabetic individuals with high genetic risk had a similar incidence of events then diabetic individuals (cumulative hazard of 33.0 versus 35.1% of diabetic subjects). The addition of combined genetic information to clinical predictors significantly improved the AUC for cardiovascular events incidence (AUC = 0.641 versus 0.610). Conclusions: Combined information of genetic variants for diabetes risk is associated to major cardiovascular events incidence, including overall mortality, in non-diabetic individuals with coronary artery disease.

An empirical evaluation of imputation accuracy for association statistics reveals increased type-I error rates in genome-wide associations

Background: Genome wide association studies (GWAS) are becoming the approach of choice to identify genetic determinants of complex phenotypes and common diseases. The astonishing amount of generated data and the use of distinct genotyping platforms with variable genomic coverage are still analytical challenges. Imputation algorithms combine directly genotyped markers information with haplotypic structure for the population of interest for the inference of a badly genotyped or missing marker and are considered a near zero cost approach to allow the comparison and combination of data generated in different studies. Several reports stated that imputed markers have an overall acceptable accuracy but no published report has performed a pair wise comparison of imputed and empiric association statistics of a complete set of GWAS markers. Results: In this report we identified a total of 73 imputed markers that yielded a nominally statistically significant association at P < 10(-5) for type 2 Diabetes Mellitus and compared them with results obtained based on empirical allelic frequencies. Interestingly, despite their overall high correlation, association statistics based on imputed frequencies were discordant in 35 of the 73 (47%) associated markers, considerably inflating the type I error rate of imputed markers. We comprehensively tested several quality thresholds, the haplotypic structure underlying imputed markers and the use of flanking markers as predictors of inaccurate association statistics derived from imputed markers. Conclusions: Our results suggest that association statistics from imputed markers showing specific MAF (Minor Allele Frequencies) range, located in weak linkage disequilibrium blocks or strongly deviating from local patterns of association are prone to have inflated false positive association signals. The present study highlights the potential of imputation procedures and proposes simple procedures for selecting the best imputed markers for follow-up genotyping studies.

CYP2C19 and ABCB1 gene polymorphisms are differently distributed according to ethnicity in the Brazilian general population

Background: Recent studies have reported the clinical importance of CYP2C19 and ABCB1 polymorphisms in an individualized approach to clopidogrel treatment. The aims of this study were to evaluate the frequencies of CYP2C19 and ABCB1 polymorphisms and to identify the clopidogrel-predicted metabolic phenotypes according to ethnic groups in a sample of individuals representative of a highly admixtured population. Methods: One hundred and eighty-three Amerindians and 1,029 subjects of the general population of 4 regions of the country were included. Genotypes for the ABCB1c.C3435T (rs1045642), CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*4 (rs28399504), CYP2C19*5 (rs56337013), and CYP2C19*17 (rs12248560) polymorphisms were detected by polymerase chain reaction followed by high resolution melting analysis. The CYP2C19*3, CYP2C19*4 and CYP2C19*5 variants were genotyped in a subsample of subjects (300 samples randomly selected). Results: The CYP2C19*3 and CYP2C19*5 variant alleles were not detected and the CYP2C19*4 variant allele presented a frequency of 0.3%. The allelic frequencies for the ABCB1c.C3435T, CYP2C19*2 and CYP2C19*17 polymorphisms were differently distributed according to ethnicity: Amerindian (51.4%, 10.4%, 15.8%); Caucasian descent (43.2%, 16.9%, 18.0%); Mulatto (35.9%, 16.5%, 21.3%); and African descent (32.8%, 20.2%, 26.3%) individuals, respectively. As a result, self-referred ethnicity was able to predict significantly different clopidogrel-predicted metabolic phenotypes prevalence even for a highly admixtured population. Conclusion: Our findings indicate the existence of inter-ethnic differences in the ABCB1 and CYP2C19 variant allele frequencies in the Brazilian general population plus Amerindians. This information could help in stratifying individuals from this population regarding clopidogrel-predicted metabolic phenotypes and design more cost-effective programs towards individualization of clopidogrel therapy.

Five-year follow-up of angiographic disease progression after medicine, angioplasty, or surgery

Background: Progression of atherosclerosis in coronary artery disease is observed through consecutive angiograms. Prognosis of this progression in patients randomized to different treatments has not been established. This study compared progression of coronary artery disease in native coronary arteries in patients undergoing surgery, angioplasty, or medical treatment. Methods: Patients (611) with stable multivessel coronary artery disease and preserved ventricular function were randomly assigned to CABG, PCI, or medical treatment alone (MT). After 5-year follow-up, 392 patients (64%) underwent new angiography. Progression was considered a new stenosis of >= 50% in an arterial segment previously considered normal or an increased grade of previous stenosis > 20% in nontreated vessels. Results: Of the 392 patients, 136 underwent CABG, 146 PCI, and 110 MT. Baseline characteristics were similar among treatment groups, except for more smokers and statin users in the MT group, more hypertensives and lower LDL-cholesterol levels in the CABG group, and more angina in the PCI group at study entry. Analysis showed greater progression in at least one native vessel in PCI patients (84%) compared with CABG (57%) and MT (74%) patients (p < 0.001). LAD coronary territory had higher progression compared with LCX and RCA (P < 0.001). PCI treatment, hypertension, male sex, and previous MI were independent risk factors for progression. No statistical difference existed between coronary events and the development of progression. Conclusion: The angioplasty treatment conferred greater progression in native coronary arteries, especially in the left anterior descending territories and treated vessels. The progression was independently associated with hypertension, male sex, and previous myocardial infarction.

Hypotheses, rationale, design, and methods for prognostic evaluation in type 2 diabetic patients with angiographically normal coronary arteries. The MASS IV-DM Trial

Background: The MASS IV-DM Trial is a large project from a single institution, the Heart Institute (InCor), University of Sao Paulo Medical School, Brazil to study ventricular function and coronary arteries in patients with type 2 diabetes mellitus. Methods/Design: The study will enroll 600 patients with type 2 diabetes who have angiographically normal ventricular function and coronary arteries. The goal of the MASS IV-DM Trial is to achieve a long-term evaluation of the development of coronary atherosclerosis by using angiograms and coronary-artery calcium scan by electron-beam computed tomography at baseline and after 5 years of follow-up. In addition, the incidence of major cardiovascular events, the dysfunction of various organs involved in this disease, particularly microalbuminuria and renal function, will be analyzed through clinical evaluation. In addition, an effort will be made to investigate in depth the presence of major cardiovascular risk factors, especially the biochemical profile, metabolic syndrome inflammatory activity, oxidative stress, endothelial function, prothrombotic factors, and profibrinolytic and platelet activity. An evaluation will be made of the polymorphism as a determinant of disease and its possible role in the genesis of micro- and macrovascular damage. Discussion: The MASS IV-DM trial is designed to include diabetic patients with clinically suspected myocardial ischemia in whom conventional angiography shows angiographically normal coronary arteries. The result of extensive investigation including angiographic follow-up by several methods, vascular reactivity, pro-thrombotic mechanisms, genetic and biochemical studies may facilitate the understanding of so-called micro- and macrovascular disease of DM.

Evaluating gene by sex and age interactions on cardiovascular risk factors in Brazilian families

Background: In family studies, it is important to evaluate the impact of genes and environmental factors on traits of interest. In particular, the relative influences of both genes and the environment may vary in different strata of the population of interest, such as young and old individuals, or males and females. Methods: In this paper, extensions of the variance components model are used to evaluate heterogeneity in the genetic and environmental variance components due to the effects of sex and age (the cutoff between young and old was 43 yrs). The data analyzed were from 81 Brazilian families (1,675 individuals) of the Baependi Family Heart Study. Results: The models allowing for heterogeneity of variance components by sex suggest that genetic and environmental variances are not different in males and females for diastolic blood pressure, LDL-cholesterol, and HDL-cholesterol, independent of the covariates included in the models. However, for systolic blood pressure, fasting glucose and triglycerides, the evidence for heterogeneity was dependent on the covariates in the model. For instance, in the presence of sex and age covariates, heterogeneity in the genetic variance component was suggested for fasting glucose. But, for systolic blood pressure, there was no evidence of heterogeneity in any of the two variance components. Except for the LDL-cholesterol, models allowing for heterogeneity by age provide evidence of heterogeneity in genetic variance for triglycerides and systolic and diastolic blood pressure. There was evidence of heterogeneity in environmental variance in fasting glucose and HDL-cholesterol. Conclusions: Our results suggest that heterogeneity in trait variances should not be ignored in the design and analyses of gene-finding studies involving these traits, as it may generate additional information about gene effects, and allow the investigation of more sophisticated models such as the model including sex-specific oligogenic variance components.

Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (Sao Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).

Spatio-Temporal Tracking and Phylodynamics of an Urban Dengue 3 Outbreak in Sao Paulo, Brazil

The dengue virus has a single-stranded positive-sense RNA genome of similar to 10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. It possesses four antigenically distinct serotypes (DENV 1-4). Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in Sao Jose do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA) were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000-2001. Sixty DENV-3 from Sao Jose do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R(0) = 1.53 and values for lineage 2 of R(0) = 1.13. Under the exponential model, TMRCA of lineage 1 dated 1 year and lineage 2 dated 3.4 years before the last sampling. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area.

The family health system: analysis of a health survey in Sao Paulo, Brazil

Background The Family Health Strategy (FHS) has been implemented as a strategy for primary care improvement in Brazil. Working with teams that include one doctor, one nurse, auxiliary nurses and community health workers in predefined areas, the FHS began in 1994 (known then as the Family Health Program) and has since grown considerably. The programme has only recently undergone assessment of outcomes, in contrast to more routine evaluations of infrastructure and process. Methods In 2001, a health survey was carried out in two administrative districts (with 190 000 inhabitants) on the outskirts of the city of Sao Paulo, both partially served by the FHS. Chronic morbidity (hypertension, diabetes and ischaemic heart disease) of individuals aged 15 or older was studied in areas covered and not covered by the programme. Stratified univariate analysis was applied for sex, age, education, income, working status and social insurance of these populations. Multivariate analysis was applied where applicable. Results There was a distinct pattern in the morbidity profile of these populations, suggesting differentiated self-knowledge on chronic disease status in the areas served by the FHS. Conclusion The FHS can increase population awareness of chronic diseases, possibly through increasing access to primary care.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Incidence and risk factors of aplastic anemia in Latin American countries: the LATIN case-control study

Background Associations between aplastic anemia and numerous drugs, pesticides and chemicals have been reported. However, at least 50% of the etiology of aplastic anemia remains unexplained. Design and Methods This was a case-control, multicenter, multinational study, designed to identify risk factors for agranulocytosis and aplastic anemia. The cases were patients with diagnosis of aplastic anemia confirmed through biopsy or bone marrow aspiration, selected through an active search of clinical laboratories, hematology clinics and medical records. The controls did not have either aplastic anemia or chronic diseases. A total of 224 patients with aplastic anemia were included in the study, each case was paired with four controls, according to sex, age group, and hospital where the case was first seen. Information was collected on demographic data, medical history, laboratory tests, medications, and other potential risk factors prior to diagnosis. Results The incidence of aplastic anemia was 1.6 cases per million per year. Higher rates of benzene exposure (>= 30 exposures per year) were associated with a greater risk of aplastic anemia (odds ratio, OR: 4.2; 95% confidence interval, CI: 1.82-9.82). Individuals exposed to chloramphenicol in the previous year had an adjusted OR for aplastic anemia of 8.7 (CI: 0.87-87.93) and those exposed to azithromycin had an adjusted OR of 11.02 (CI 1.14-108.02). Conclusions The incidence of aplastic anemia in Latin America countries is low. Although the research study centers had a high coverage of health services, the underreporting of cases of aplastic anemia in selected regions can be discussed. Frequent exposure to benzene-based products increases the risk for aplastic anemia. Few associations with specific drugs were found, and it is likely that some of these were due to chance alone.

Long-acting injectable risperidone in partially adherent and nonadherent patients with schizophrenia

Background: Long-acting injectable antipsychotics may improve medication adherence, thereby improving overall treatment effectiveness. This study aimed to evaluate the effectiveness, safety, and tolerability of risperidone long-acting injection in schizophrenic patients switched from oral antipsychotic medication. Methods: In a 12-month, multicenter, open-label, noncomparative study, symptomatically stable patients on oral antipsychotic medication with poor treatment adherence during the previous 12 months received intramuscular injections of risperidone long-acting injection (25 mg starting dose) every 2 weeks. The primary endpoint was the change in Positive and Negative Syndrome Scale (PANSS) total score. Results: Of the 60 patients who were screened, 53 received at least one injection (safety population), and 51 provided at least one postbaseline assessment. Mean PANSS total scores improved significantly throughout the study and at endpoint. Significant improvements were also observed in Clinical Global Impression of Severity, Personal and Social Performance, and Drug Attitude Inventory scales. Risperidone long-acting injection was safe and well-tolerated. Severity of movement disorders on the Extrapyramidal Symptom Rating Scale was reduced significantly. The most frequently reported adverse events were insomnia (22.6%), increased prolactin (17.0%), and weight gain (13.2%). Conclusion: Risperidone long-acting injection was associated with significant symptomatic improvements in stable patients with schizophrenia following a switch from previous antipsychotic medications.